Molecular detection of genotypic clarithromycin‐resistant strains and its effect on the eradication rate of concomitant therapy in Helicobacter pylori infection

Antimicrobial eradication rates for Helicobacter pylori have been decreasing and the reason for treatment failure was found to be resistance to one or more of the antibiotics. Clarithromycin resistance to H pylori was associated with point mutations in the 23S rRNA gene and the PCR‐RFLP method can detect these point mutations. The aim of this study was to determine the molecular detection of genotypic clarithromycin‐resistant strains and its effect on the eradication rate of concomitant therapy in H pylori infection. The presence of H pylori DNA was confirmed by amplifying the UreC gene by polymerase chain reaction (PCR) and point mutations on 23S rRNA (A2142G and A2143G) were detected by PCR‐RFLP. A total of 98 H pylori‐infected patients were involved and among them, genotypic clarithromycin‐sensitive strain was 93.9% and clarithromycin‐resistant strain was 6.1%. All patients were found to have the A2143G point mutation but A2142G was not detected. Successful eradication rate of concomitant therapy was found to be 89.8% and unsuccessful rate was 10.2%. Among patients with the clarithromycin‐resistant gene, only 16.7% had successful eradication and 83.3% had unsuccessful eradication. There was a statistically significant association between failure rate of concomitant therapy and detection of clarithromycin‐resistant genes (P < 0.01). The presence of A2143G point mutation in the clarithromycin‐resistant strain has a negative effect on the eradication rate of H pylori infection.


| INTRODUC TI ON
Helicobacter pylori infection, the most common chronic bacterial infection in the world, is linked to peptic ulcer disease and gastric cancer and is a key constituent of the human microbiome. They are unique bacteria ideally suited to live within the acidic environment of the human stomach, their spiral shape and multiple unipolar flagella allow them to manoeuvre freely through the gastric mucous layer in a microenvironment protected from low gastric pH. 1 Among South-East Asian countries, the reported seroprevalence rate in volunteer blood donor was 35.9% in Malaysia, 31% in Singapore and 57% in Thailand. 2 In Myanmar, the prevalence of H pylori infection among asymptomatic Buddhist monks was 65.4% by urea breath test, 3 78.6% in dyspeptic patients by rapid urease test and histology, 4 77.6% in dyspeptic patients by rapid urease test only 5 and 30% by culture, 6 respectively. One of the community studies reported that the overall seroprevalence of H pylori was 68.8%. 7 The antimicrobial eradication rates for H pylori have been decreasing and the most likely primary reason for treatment failure were found to be H pylori resistance to one or more of the antibiotics and patient compliance. 8 The rates of antibiotic resistance for H pylori are increasing throughout the world and they vary geographically and are higher in developing countries. 9 Treatment success rates can vary among countries and regionally within countries, related to antibiotic resistance and local ecology. 10 The variability of the clarithromycin-resistant rate of H pylori seen in several regions emphasises the necessity to look at resistance rates in each geographical area to better guide treatment regimens. World Health Organization recently announced that the emergence of clarithromycin-resistant strains can cause global H pylori treatment failure. 11 In ASEAN countries the prevalence of clarithromycin resistance rate varied from 2% to over 30%. 12 The efficacy of current conventional clarithromycin-based triple therapy has decreased to an unacceptably low level worldwide (lower than 80%). 8 An earlier study found that the clarithromycin resistance rate in Myanmar was 12.5% 6 and a second study in 2015 showed an increased to 63.6% by using the Episilometer test in Myanmar. 13 In a recent intention-to-treat analysis, the eradication rate of both clarithromycin and levofloxacin was unacceptably low at 40% and 43.7%, respectively. 14 Therefore, quadruple therapy is the method of choice for H pylori eradication in Myanmar. As bismuth is locally not available, we used non-bismuth quadruple therapy (concomitant). The eradication rate of concomitant therapy was 92.2% in Myanmar, 15 77% in India 16 and 91.7% in Spain. 17 Resistance to clarithromycin, an antibiotic in the treatment of H pylori infection, is due to a lack of binding of the drug to the 23S rRNA of the bacterial ribosome, which is caused by the occurrence of point mutations in the variable domains of the peptidyl transferase of 23S rRNA. Point mutations in the 23S rRNA gene, mainly at positions 2142 and 2143 with a transition of A to G, are responsible for the resistance. 18 The basic assay, first described in 1996, utilises a PCR-RFLP approach in which the region of the gene containing the mutations is amplified and then digested with restriction endonucleases that cut specifically at the mutation sites. 19 A significant development was the invention that the PCR-RFLP assay might be successfully applied to the evaluation of clarithromycin resistance without culture by direct analysis of DNA extracted from gastric biopsies. 20 Its culture, as well as antimicrobial susceptibility studies are difficult to perform as well as labour intensive.
Although the clarithromycin-resistant rate was variable in Myanmar, its effect on eradication rate of H pylori infection has not been evaluated. This study set out to determine the molecular detection of genotypic clarithromycin resistance strains and its effect on the eradication rate of concomitant therapy in H pylori infection.
This study may have the potential to provide data for national survey for H pylori infection.

| MATERIAL S AND ME THODS
This hospital-based cross-sectional analytic study was conducted between October 2019 and October 2020 at the Gastroenterology Unit, No. (2) Military Hospital (500-bedded) and the Defence Services Medical Academy, Yangon. Inclusion criteria were patients who were found to have H pylori infection detected by rapid urease test, age more than 18 years and both sexes. Patients who had taken proton pump inhibitors in the prior 2 weeks, antibiotics and bismuth in the prior 4 weeks, allergy to amoxicillin, tinidazole and clarithromycin and those who did not give informed consent were excluded.

| Patient enrolment
Dyspeptic patients indicated for upper GI endoscopy who attended the outpatient clinic or admitted to the Gastroenterology Department were recruited. Two pieces of gastric mucosa from the antrum and the middle of the body were taken for detection of H pylori by rapid urease test (proton dry test) from all enrolled patients.
The tissue samples were put into a Pronto Dry well. The result was obtained after 20 minutes. An additional one-piece tissue biopsy from the antrum was also taken for detecting the clarithromycinresistant gene by PCR-RFLP. If the patient with RUT test was negative, he or she was excluded from the study and the biopsy sample for molecular analysis was discarded according to the standard procedure of the Gastroenterology Department. If RUT turned out to be positive, biopsy for clarithromycin-resistant gene by RFLP was stored at −20°C in a freezer at the Post Graduate Common Research Laboratory. DNA was extracted within 12 hours followed by molecular tests for detection of the clarithromycin-resistant gene.

| DNA extraction and genotyping
The DNA was extracted from biopsy specimens from the gastric antrum using a PureLink Genomic DNA Mini Kit (Invitrogen) and DNA quality and quantity were checked by gel electrophoresis and a Nanodrop spectrophotometer (BioSpec Nano).

| Helicobacter pylori eradication therapy
All H pylori-positive patients were treated with concomitant therapy (amoxicillin 1G, clarithromycin 500 mg, tinidazole 500 mg, pantoprazole 40 mg, all given twice daily) for 14 days. The patients were offered medication and explained about the dose, timing and duration of all prescribed drugs. They were trained to write whether or not they took their prescribed medications and any adverse events they experienced. Patients were asked to visit the investigator on days and 15. Thereafter, resolution of presenting symptoms and drugrelated adverse effects were assessed and compliance was checked by calculating pill count. At the 4th week after H pylori therapy, successful H pylori eradication was detected by stool antigen test. Rapid urease test was not used for confirmation of H pylori eradication in our study because not every patient was not indicated to do recheck endoscopy.

| Data analysis
Data were analysed using ibm spss version 22. The Fisher's exact tests were calculated for finding the associations between clarithromycinsensitive and -resistant H pylori strains, age, sex and endoscopic findings. The chi-squared test was used for comparing the eradication rates in clarithromycin-sensitive and -resistant H pylori strains after giving concomitant therapy.

| Ethical considerations
Ethical clearance was obtained from the Ethical Committee of the Defence Services Medical Academy.

| RE SULTS
A total of 171 patients ( Figure 1)

| CON CLUS ION
In the present research, the rate of clarithromycin-resistant H py lori genotype was low and was mainly associated with the A2143G point mutation. However, A2142G point mutation was not detected.
According to our results, clarithromycin-containing H pylori eradication therapy might still be recommended as empirical therapy in Myanmar.

ACK N OWLED G EM ENT
We would like to express our heartfelt thanks to all the members of the board of post-graduate medical study giving us the opportunity to undertake this study and also like to thank all the patients for their participation and cooperation in this study.
Declaration of personal interests: None.

AUTH O R S H I P
Guarantor of the article: None.
Author contributions: None.

E TH I C A L S TATEM ENT
The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to and ethical approval was obtained from the Ethics Review Committee of the Defence Services Medical Academy.

PE E R R E V I E W
The peer review history for this article is available at https://publo ns.com/publo n/10.1002/ygh2.476.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.